Structure of the human pancreatic cholesterol esterase gene.

The gene for human pancreatic cholesterol esterase consists of 11 exons and 10 introns and is 9.2 kb in length. The last and longest exon (841 nucleotides) is unique to the human gene. Functional amino acids are encoded on separate exons. The leader sequence is encoded by a single exon which carries two additional N-terminal amino acids of the mature functional protein. A positive TATA element is identified 43 nucleotides from the start codon. Pulse-field gel electrophoresis and hybridization with various cDNA probes and direct sequence data revealed the existence of a CEase-like gene. Partial sequence analysis of this gene from a human cosmid library and human genomic DNA showed a premature stop signal in exon 10, shortly after the codon for the active-site histidine. Both the functional gene and the CEase-like gene have a polyadenylation signal in the 3'-untranslated region. Thus, the complex gene structure for this intestinally active enzyme may provide in part a potential molecular explanation for the well-known heterogeneity of the intestinal absorption of cholesterol.     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

A cluster of alpha 2-macroglobulin-related genes (alpha 2 M) on human chromosome 12p: cloning of the pregnancy-zone protein gene and an alpha 2M pseudogene

The characterization of two alpha 2-macroglobulin (alpha 2M)-related genomic clones, isolated from two human genomic libraries by use of alpha 2M cDNA [Kan et al., Proc. Natl. Acad. Sci. USA 82 (1985) 2282-2286] as a probe, is reported. Sequence comparison of the clone EPZP6 with the human alpha 2M cDNA revealed the presence of five exons with the proper splice signals. Alignment of the corresponding amino acid (aa) sequence of these exons with the published partial pregnancy-zone protein (PZP) aa sequence (Sottrup-Jensen et al., Proc. Natl. Acad. Sci. USA 81 (1984) 7353-7357] showed a perfect match, thereby identifying EPZP6 as a PZP genomic clone. The clone MPAM16 showed a considerable degree of sequence conservation when compared to the human alpha 2M cDNA sequence, and several putative exons were identified. However, a frame-shift mutation leading to a premature stop codon was found in the coding sequence, classifying this gene as an alpha 2M pseudogene. Human alpha 2M, PZP and the related pseudogene were mapped to the human chromosome 12p12-13, with the help of gene-specific probes and in situ hybridization. This result was confirmed in Southern-blot experiments with DNA from a human-Ltk- mouse somatic-cell hybrid containing only a human isochromosome 12p in a mouse background.     

Guinea pigs possess a highly mutated gene for L-gulono-gamma-lactone oxidase, the key enzyme for L-ascorbic acid biosynthesis missing in this species.

Guinea pigs cannot synthesize L-ascorbic acid because of their deficiency in L-gulono-gamma-lactone oxidase, a key enzyme for the biosynthesis of this vitamin in higher animals. In this study we isolated the L-gulono-gamma-lactone oxidase gene of the rat and the homologue of this gene of the guinea pig by screening rat and guinea pig genomic DNA libraries in lambda phage vectors, respectively, using a rat L-gulono-gamma-lactone oxidase cDNA as a probe. Sequencing analysis showed that the amino acid sequence of the rat enzyme is encoded by 12 exons and that all the intron/exon boundaries follow the GT/AG rule. On the other hand, regions corresponding to exons I and V were not identified in the guinea pig L-gulono-gamma-lactone oxidase gene homologue. Other defects found in this gene homologue are a deletion of the nucleotide sequence corresponding to a 3' 84-base pair part of rat exon VI, a 2-base pair deletion in the remaining exon VI-related region, and nonconformance to the GT/AG rule at one of the putative intron/exon boundaries. Furthermore, a large number of mutations were found in the amino acid-coding regions of the guinea pig sequence; more than half of them lead to nonconservative amino acid changes, and there are three stop codons as well. Thus it is clear that the guinea pig homologue of the L-gulono-gamma-lactone oxidase gene exists as a pseudogene that randomly accumulated a large number of mutations without functional constraint since the gene ceased to be active during evolution. On the basis of the neutral theory of evolution, the date of the loss of L-gulono-gamma-lactone oxidase in the ancestors of the guinea pig was roughly calculated to be less than 20 million years ago.     

Molecular and genetic analysis of a compound heterozygote for dysprothrombinemia of prothrombin Tokushima and hypoprothrombinemia.

The molecular and genetic basis of a compound heterozygote for dys- and hypoprothrombinemia was analyzed. Abnormal nucleotide sequences of the human prothrombin gene were screened by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion and mutated primer-mediated PCR-RFLP. A single nucleotide substitution responsible for dysprothrombinemia of prothrombin Tokushima was detected, as were three polymorphisms. The mutation for hypoprothrombinemia was detected by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion in exon 6, near MboII-RFLP and NcoI-RFLP. Sequencing of PCR-amplified genomic DNA revealed a single base insertion of thymine (T) at position 4177. The resulting frameshift mutation caused both an altered amino acid sequence from codon 114 and a premature termination codon (i.e., TGA) at codon 174 in exon 7. Because exon 7 encodes the kringle 2 domain preceding the thrombin sequence, this frameshift leads to the null prothrombin phenotype. The inheritance of the hypoprothrombinemia gene from the father to the proband was proved by PCR-SSCP with endonuclease digestion and mutated primer-mediated PCR-RFLP.     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

Molecular basis of a selective C1s deficiency associated with early onset multiple autoimmune diseases

We have investigated the molecular basis of selective and complete C1s deficiency in 2-year-old girl with complex autoimmune diseases including lupus-like syndrome, Hashimoto's thyroiditis, and autoimmune hepatitis. This patient's complement profile was characterized by the absence of CH50 activity, C1 functional activity <10%, and undetectable levels of C1s Ag associated with normal levels of C1r and C1q Ags. Exon-specific amplification of genomic DNA by PCR followed by direct sequence analysis revealed a homozygous nonsense mutation in the C1s gene exon XII at codon 534, caused by a nucleotide substitution from C (CGA for arginine) to T (TGA for stop codon). Both parents were heterozygous for this mutation. We used the new restriction site for endonuclease Fok-1 created by the mutation to detect this mutation in the genomic DNA of seven healthy family members. Four additional heterozygotes for the mutation were identified in two generations. Our data characterize for the first time the genetic defect of a selective and complete C1s deficiency in a Caucasian patient.     

Seven novel Tay-Sachs mutations detected by chemical mismatch cleavage of PCR-amplified cDNA fragments.

Total RNA was isolated from cultured fibroblasts from 12 unrelated patients with Tay-Sachs disease, an autosomal recessive disorder due to beta-hexosaminidase A deficiency. beta-Hexosaminidase mRNA was amplified by cDNA-PCR in four overlapping segments spanning the entire coding sequence. In two patients, abnormal size cDNA-PCR fragments in which exons were removed resulted from splicing mutations that were characterized at the genomic DNA level: both were G to A transitions, at the first position of intron 2 and at the fifth position of intron 4. Five other mutations have been identified by cDNA-PCR chemical mismatch analysis and direct sequencing of an amplified fragment containing the mismatch site. One missense mutation alters the codon for Ser210 to Phe in exon 6 and the other one alters the codon for Arg504 to Cys in exon 13. A 3-bp deletion results in the deletion of a phenylalanine residue in exon 8. Two nonsense mutations in exon 3 (Arg137 to stop) and in exon 11 (Arg393 to stop) are associated with a marked decrease of mRNA abundance, probably because they result in mRNA instability. Three of the six single base mutations involve the conversion of a CpG dinucleotide in the sense strand to TpG. These results demonstrate the extreme molecular heterogeneity of mutations causing Tay-Sachs disease. The procedure described in this paper allows the rapid detection of any type of mutation, except those impairing the promoter function. Applicable even to patients with splicing or nonsense mutations and very low mRNA abundance, it has therefore a potentially broad application in human genetics, for both diagnostic and fundamental purposes.     

Elements of the rat tropoelastin gene associated with alternative splicing

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.     

Computational and experimental analyses reveal previously undetected coding exons of the KRIT1 (CCM1) gene

A notable difficulty in annotating genomic sequence is identifying the correct start codon in a gene. An important such case has been found with KRIT1, the cerebral cavernous malformation type 1 (CCM1) gene. Analysis of human and mouse genomic sequence encompassing the region containing KRIT1/Krit1 using exon/gene-prediction and comparative alignment programs revealed putative exons upstream of the previously described first exon. These additional candidate exons show significant matches to mouse and human ESTs that are contiguous with and extend upstream from the previously designated 5' end of the KRIT1 cDNA sequence. RT-PCR and 5'RACE experiments confirm the presence of four additional upstream coding exons that encode an additional 207 amino acids. Importantly, a novel frameshift mutation in one of these newly identified KRIT1 exons has been found in a CCM1 family. These data establish the authentic KRIT1 amino acid sequence and suggest that the additional KRIT1 exons may harbor mutations in other CCM1 families. In addition, these results provide another example of the utility of rigorous computational and comparative sequence analysis for refining gene structure.     

Organization of the murine Ig-related lambda 5 gene transcribed selectively in pre-B lymphocytes.

lambda 5 is an immunoglobulin lambda light chain-related gene which is selectively transcribed in murine pre-B lymphocytes to yield a 1.2 kb poly(A)+ mRNA. Comparison of the nucleotide sequence of a 1 kb cDNA clone with the sequence of a genomic clone isolated from 70Z/3 murine pre-B lymphoma cells shows lambda 5 is composed of three exons spanning a 3.75 kb DNA segment. Conserved splice signal sequences at all exon/intron boundaries and the presence of a long open reading frame indicate that a functional mRNA molecule can be made. Exon I contains a cap-site and a potential ATG start codon as well as sequences encoding a signal peptide. This gene could encode a lambda 5 protein of 209 amino acids which has, however, not yet been identified. The 3' portion of exon II and all of exon III shows strong sequence homologies to J lambda L and C lambda L exons. Homology to the lambda L chain genes is lost in the 5' portion of exon II and throughout exon I. In exon I short homologies to leader sequences and to VH framework 1 sequences are seen.     

Partial characterization of bovine elastin gene; comparison with the gene for human elastin

A 14 kilobase (kb) genomic clone of the gene for bovine elastin, containing exons 1 and 2, has been characterized. This clone extends about 6.5 kb in the 5' direction from the initiation codon and 978 nucleotides in the 3' direction from exon 2. The size of the first intron is about 6.4 kb. The sequence immediately 5' to the initiation codon is highly conserved between the genes for bovine and human elastins and contains a TATA box consensus sequence (ATAAA), CAAT, and Sp1 binding sites. Several putative AP-2 binding sites are also present. Comparative analysis of the sequences flanking the first exon in the genes for bovine and human elastins identified conserved sequences that may be regulatory control elements. A putative enhancer core sequence is present in the first intron of the genes for bovine and human elastins.     

Deletion within the CYP17 gene together with insertion of foreign DNA is the cause of combined complete 17 alpha-hydroxylase/17,20-lyase deficiency in an Italian patient.

The molecular basis of 17 alpha-hydroxylase/17,20-lyase deficiency syndrome in a 14-yr-old 46,XY Italian patient was investigated by amplification, subcloning, and sequencing of specific exonic sequences from genomic DNA samples. A homozygous mutation, consisting of a 518-basepair (bp) deletion combined with a 469-bp insertion, was identified in the CYP17 gene of the patient. The deletion spans much of exon II, the whole intron 2, and a portion of exon III. A part (156 bp) of the inserted sequence shows 95.5% identity to the nuclear antigen-binding site on Marek disease virus DNA and sequences found in rearranged mitochondrial DNA of rat hepatoma cells. A similar degree of sequence identity (99%) was also found between the above sequences and part of the lac operon of E. coli. The inserted sequence is lacking the BamHI site in intron 2 of CYP17 and contains an in-frame stop codon (TAA). Thus, the mutated gene encodes a truncated nonfunctional steroid hydroxylase, giving rise to symptoms associated with complete combined 17 alpha-hydroxylase/17,20-lyase deficiency. The family history revealed that the patient is the child of a consanguineous marriage and has two genotypically and phenotypically female sisters also suffering from symptoms of the disease. Investigation of genomic DNA from these sisters revealed that in each case both CYP17 alleles contained the same mutation. On the other hand, the parents were found to be heterozygous for this mutation. The insertion could not be found in DNA from normal individuals or in the CYP17 gene of other Italian patients with the 17 alpha-hydroxylase deficiency syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)